Field of the Invention
The present invention relates to the field of nanodevices for single cell extraction of intracellular contents, specifically, mitochondrial DNA.
Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual compositions or methods used in the present invention may be described in greater detail in the publications and patents discussed below, which may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance or the prior art effect of the patents or publications described.
Physiological and pathological processes within the human body are controlled by complex cell-cell interactions within the context of a dynamic microenvironment. Biomolecular analysis of cells has been traditionally conducted under the assumption that cells of a clonal population behave identically. This assumption arose not from experimental evidence but from the lack of tools capable of analyzing individual cells. Furthermore, the ability to dynamically measure phenotypes (i.e. gene expression, protein activities, ion fluctuations, signaling) at the single cell level is key to understanding cellular behavior in a complex environment (1-4).
In 1990, James Eberwine's group isolated a single-cell in culture using a glass micropipette and demonstrated that RNA extracted from that cell could be amplified and analyzed (5). This experiment pioneered the field of single-cell biology and Eberwine's group successfully applied this technology towards understanding the molecular basis of neuronal functioning (6). In the last 20 years, biomolecular analysis leaped forward with the development of high-throughput sequencing which now allows researchers to obtain the collection of active genes in a cell in a single readout (7). The initial capture of the single cell, however, remains a major technical issue (1). Since Eberwine's success using a glass micropipette, scientists have used a variety of techniques to isolate single cells, from enzymatic digestion (which releases cells from tissues) to laser micro-dissection (which cuts out a homogeneous population of intact cells from tissue sections). However, these methods either cannot examine cells in their native environment, or as with the original micropipette, they are limited to probing isolated cells (8).
Nanodevices as Surgical Tools
Nanoscale devices are ideal single-cell surgical tools because of their potential for high spatial and temporal resolution studies (9-10). Recently two groups independently developed cellular nanoendoscopes for single cell analysis. Singhal et al. (11) attached a carbon nanotube at the very tip of a glass micropipette and showed its potential for interrogating cells, transporting fluids and performing optical and electrochemical diagnostics down to the single organelle level. Similarly Yan et al. (12) developed a nanowire waveguide attached to the tip of an optical fiber which can both guide visible light into intracellular compartments of a living mammalian cell and detect optical signals from sub-cellular regions. The cylindrical shape of these nanoendoscopes allows the probing of organelles deep inside the cell but these technologies lack any automation capability; these endoscopes are manually positioned in the cell, not allowing the study of those complex biological problems that require analysis of a large number of samples.
Integration of these nanoendoscopes with scanning probe techniques may overcome this limitation, potentially allowing automation and high-throughput analysis. In 2003, Osada and coworkers inserted atomic force microscope (AFM) tips into living cells to extract mRNAs (13). The mRNAs were analyzed with PCR techniques to validate the extraction protocol (14). More recently, Wickramasinghe's group optimized this method by coating AFM tips with platinum, which allowed extraction of the mRNA molecules through dielectrophoresis. Wichramasinghe's technology has been successfully combined with standard assay techniques to detect RNA molecules in breast cancer cells (15-16).
Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. With the introduction of high-throughput sequencing it is now possible to obtain the collection of all active genes within a single cell, but the initial capture of genetic material of the cell still remains a major challenge. Furthermore, current methods for single-cell manipulation often can detect only one class of analytes.